PROJECT SUMMARY The presentation of self and foreign antigens to T lymphocytes involves a variety of cellular processes, including proteolysis, endocytosis, phagocytosis, vesicle trafficking and autophagy. Our recent studies have investigated the role of autophagy, a self-degradation mechanism, and related cellular processes in MHC class I- and class II-restricted antigen presentation to T lymphocytes, with the long-term goal of manipulating these pathways to prevent or treat human disease. We have focused on a key player in autophagy, the class III phosphatidylinositol- 3 kinase (PI3K) vacuolar protein sorting 34 (Vps34). Our published and unpublished preliminary studies have provided evidence for a key role of Vps34 in both MHC class I- and class II-restricted antigen presentation by dendritic cells (DCs), which are critical players in the induction of tolerance against self-antigens and for initiating adaptive immune responses against foreign antigens. We have shown that the classical MHC class I and class II antigen presentation pathways are enhanced in Vps34-deficient DCs, and that these cells harbor a specific defect in the cross-presentation of apoptotic cell-associated antigens to MHC class I-restricted CD8+ T cells. Moreover, using mice carrying a selective deficiency of Vps34 in their thymic epithelial cells (TECs), we have shown a critical role of Vps34-mediated cellular processes in intrathymic T lymphocyte development. Guided by this scientific premise we propose the overall hypothesis that Vps34-mediated, autophagy-related processes play critical roles in presenting antigens to T lymphocytes and promoting self-tolerance. We will test this hypothesis in the following specific aims: Aim 1 will investigate the contribution of Vps34-mediated functions in unconventional MHC class I- and class II-restricted antigen presentation, and will explore the autophagy-dependent and -independent molecular mechanisms involved. Aim 2 will interrogate the role of Vps34 in the intrathymic development and selection of the T cell repertoire. Aim 3 will explore the role of Vps34 in maintaining peripheral tolerance and controlling T lymphocyte responses against autoantigens. In these studies, we will employ a variety of experimental approaches, including in vitro and in vivo antigen presentation assays, and various genetically engineered animals. As global Vps34 gene-deficiency is incompatible with life, we will employ mice selectively deficient in Vps34 expression in either DCs or TECs. We will complement these studies with cultures of murine and human cells using pharmacological modulators of Vps34 function. We will also take advantage of a variety of reporter animals and transgenic mice expressing neo self-antigens and/or antigen- specific T cell receptors. Collectively, these proposed studies will provide us with a comprehensive mechanistic view of the contribution of the autophagy-related protein Vps34 in antigen presentation, T cell development and selection, peripheral tolerance and T cell immune responsiveness. These proposed studies will be instrumental for the development of improved vaccines and immunotherapies against microbial pathogens and tumors, and for devising methods to induce tolerance against self or foreign antigens.